1. Field of the Invention
The object of the invention are amino acid sequences (peptides) produced from antibody producing cells, particularly monoclonal antibody producing hybridoma cells, that neutralize the effect of the Clostridium difficile enterotoxin and/or cytotoxin. In addition, there is a description of humanized, monoclonal antibodies for use against the Clostridium difficile toxins, as well as the hypervariable regions of these antibodies. Finally a process for the production and application of these amino acid sequences (peptides) and monoclonal antibodies is shown.
2. Description of the Related Art
It is known that the introduction of macrolide antibiotics such as Clindamycin leads to severe bowel disease, which manifests as diarrhoea, proceeding further to occasionally fatal pseudomembranous colitis (PMC). This connection gave the disease the name “Clindamycin associated diarrhea.” We now know that nearly all the antibiotics and cytostatic agents used in medicine can trigger the clinical symptoms of PMC.
PMC is characterized clinically by severe diarrhoea that may lead to death due to heavy electrolyte and liquid losses. Depending on the severity of the symptoms, abdominal pain, bloody diarrhoea, fever and leukocytosis occur. Treatment has hitherto consisted in stopping of the administration of the antibiotic causing the disease, by the administration of Vancomycin, as well as balancing liquid and electrolyte losses.
The aetiological agent of pseudomembranous colitis was for a long time unknown. Only in 1977 could hitherto unknown toxic activity be demonstrated in a stool specimen, inducing a cytotoxic effect on CHO cells (Chinese hamster ovary carcinoma cells). Through further investigations it could finally be proved that the pseudomembranous colitis was caused by Clostridium difficile and its toxins.
Clostridium difficile is an obligate anaerobic, gram-positive rod bacteria that builds subterminal oval spores. It is characterized biochemically by being able to ferment monosaccharides such as glucose, N-acetylglucosamine and N-acetylneuraminic acid, but not mannose, xylose or arabinose. Clostridium difficile is also not able to split these monosaccharides off from the side chains of the gastrointestinal mucin as enzymes such as neuraminidase, beta-galactosidase or sialidase are missing. Due to these biochemical shortcomings Clostridium difficile cannot flourish in the stomach of healthy individuals. If the gut flora are disturbed through the administration of antibiotics or cytostatic agents, Clostridium difficile outgrows the gut flora, leading to PMC.
Clostridium difficile produces two major factors of pathogenicity, the enterotoxin (toxin A) and the cytotoxin (toxin B). Their production, purification and properties, together with their use to produce monoclonal antibodies are described in detail in European patents 153 519 and 209 273, U.S. Pat. Nos. 4,879,218 and 5,098,826 as well as international patent application WO 91/18 293.
Antibodies clearly play an important part in protecting against the consequences of infection with Clostridium difficile. Antibody titers against toxin A and toxin B could be established in patients suffering from PMC. After antibiotic treatment and infection with toxinogenic Clostridium difficile strains, hamsters develop the symptoms of PMC and die of it. Prior immunization of the animals with the above-mentioned toxins protects them from disease. The toxins can be neutralized by antibodies, which are directed against the C-terminal repetitive ligand domains, the central translocation domain or the N-terminal catalytic domain (see reference [1] of the bibliography regarding domain structure). The antibodies directed against the C-terminal repetitive ligand domains hinder the binding of the toxins to the cell receptors, the antibodies directed against the N-terminal catalytic domain block the glucosylation reaction imparted by the toxins, while the antibodies directed against the central translocation domain restrain the translocation of toxins into the cell.
Antibodies neutralizing toxin A and/or toxin B thus offer a possible therapy and/or prophylaxis for Clostridium difficile diseases in which they do not eliminate the bacteria but they block the action of the toxins produced. In this way the development of the symptoms of the disease can be hindered by acting on the toxins responsible.